The trefoil factor family consists of 3 proteins: pS2/TFF1, spasmolytic polypeptide (SP/TFF2) and intestinal trefoil factor (ITF/TFF3), which play an essential role in the homeostasis of gastrointestinal (Gl) epithelial cell surfaces. The 3 TFFs share many structural and functional similarities. Functionally, each has been shown to promote epithelial healing by enhancing cell migration while preventing apoptosis. Structurally, these small proteins share an absolutely conserved motif (the "trefoil domain") that forms a three-loop structure by virtue of disulphide linkages between the six cysteine residues. Despite considerable structural homology, TFF1 and TFF3 appear to play different roles in cancer cells. TFF1 has tumor suppressor activity in the stomach: TFF1 knockout mice develop gastric adenomas and carcinomas, and in human gastric cancer, TFF1 loss occurs by gene mutations, loss of heterozygosity, and methylation silencing. We recently demonstrated that gastric cancer-associated mutations of TFF1 lose their tumor suppressor function and actually enhance cancer cell invasion by distinct signaling pathways. We also found that ectopic expression of TFF3 by human gastric cancers is associated with a poor prognosis, independent of tumor stage, and that transfecting TFF3 into TFF3(-) non-aggressive rat colon cancer cells converts them to a more aggressive phenotype. The overall goal of the present proposal is to better understand how the structurally related TFF1, mutant TFF1, and TFF3 peptides can have rather disparate functional effects on Gl cancer cells. Our hypothesis is that specific structural elements of TFF1 and TFF3 are responsible for the different functions of these peptides. The following Specific Aims will be explored: (1) Determine how TFF3 contributes to a more malignant phenotype in cancer cells;(2) Determine how TFF1 confers its tumor suppressor function;(3) Define key signaling pathways used by TFF1, mutant TFF1, and TFF3 during cancer cell invasion. The results of these studies could provide insights into novel treatment strategies for gastrointestinal malignancies.